Quantitative Proteomics Reveals Significant Differences Between Mouse Brain Formations

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Abstract: Aging is associated with a general decline in cognitive functions, which appears to be due to alterations in the amounts of proteins involved in the regulation of synaptic plasticity. Here, we present a quantitative analysis of proteins involved in neurotransmission in three brain regions, namely, the hippocampus, the cerebral cortex, and the cerebellum, in mice aged 1 and 22 months, using the total protein approach technique. We dernonstrate that although the titer of some proteins involved in neurotransmission and synaptic plasticity is affected by aging in a similar manner in all the studied brain formations, in fact, each of the formations represents its own mode of aging. Generally the hippocampal and cortical proteomes are much more unstable during the lifetime than the cerebellar proteome. The data presented here provide a general picture of the effect of physiological aging on synaptic plasticity and might suggest potential drug targets for anti-aging therapies.

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Keywords: glutamatergic and GABAergic transmission; Camk2; OXPHOS; extracellular matrix; total protein approach;hippocampus;cortex;cerebellum

1. Introduction

Physiological aging is related to a gradual decline in cognitive functions, such as memory formation and retention, processing speed and conceptual reasoning [1]. It is widely accepted that aging-associated changes are caused mainly by the loss of neuronal cells; however, numerous studies have suggested that the neuronal network structure, rather than the number of neurons, is affected during aging [2-4]. It has been demonstrated that aging is associated with the shortening ofdendrites and a decrease in their number, loss of dendritic spines, a decrease in the axon number within a network and the level of their myelination and loss of synapses [5]. Additionally, it has been shown that the molecular mechanisms underlying brain plasticity phenomena such as long-term potentiation and depression of the glutamatergic (LTP and LTD) and GABAergic (iLTP and iLTD) synapses and the efficacy of various neuromodulator systems decline with advancing age [6,7]. Several studies have demonstrated that aging-related cognitive changes are accompanied by changes in the expression and/or localization of proteins involved in synaptic transmission and plasticity [6,8]. The expression of proteins in various regions of the rodent brain has been intensively studied by mass spectrometry-based proteomic techniques[9,10]and microarrays[11,12]. cistanche cholesterol However, except for Walter and Mann's study [10] and Duda et al.'s study [8],all the investigations employed semiquantitative approaches which do not deliver information about the accurate concentration of proteins in the studied samples.

Recently, we measured concentrations of proteins involved in neuronal plasticity in the hippocampus, cerebral cortex and cerebellum in young (1-month-old) and adult(12-month--old) mice [8]. We found that, while the total amount of proteins did not change during the lifetime, the neurotransmission- and neuroplasticity-related protein titers differed significantly between young and adult animals, which indicates that the symptoms of signal transmission and neuroplasticity weakening may be observed in middle-aged mice at a proteomic level [8].

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In this paper, we provide the results of an in-depth quantitative analysis of neuronal plasticity-related hippocampal, cortical, and cerebellar proteomes of young (1-month-old)and old (22-month-old) mice, focusing mainly on aging-related changes in the concentration of proteins engaged in glutamate, GABA, acetylcholine and monoamine signaling. We also discuss the changes in the concentration of proteins involved in neurotransmitter release and formation of synaptic connections, such as proteins of trans-synaptic cell adhesion and perineuronal nets.

Using the label-free total protein approach (TPA)method, we measured the titers of more than 7000 proteins in each of the studied brain regions [13]. In this paper, we present, thus far, the most in-depth quantitative proteomic description of synaptic plasticity-related changes during the physiological aging of mice. To the best of our knowledge, this is, thus far, the most in-depth quantitative proteomic description of synaptic plasticity-related changes during the physiological aging of mice.

2. Materials and Methods

2.1. Animals and Preparation of Tissues

Brains were isolated from five female C57BL/10J mice at P30 (young) and five 22-month-old (aged)mice. Animals were treated as described in [8]. Briefly, the animals were anes-thetized with isoflurane and decapitated, and brains were explanted in an ice-cold buffer (87mMNaCl,2.5mMKCl,1.25mMNaHNPO4,25mMNaHCO3,0.5mMCaClz,7mMMgSO, 25mM glucose,75 mM sucrose, pH7.4). The whole brain regions: right hippocampus, frontal cortex from the right hemisphere and right hemisphere of the cerebellum, from each animal, were used for quantitative proteomics. cistanche deserticola side effects All the procedures were approved by the local ethics committee (Wroclaw Ethical Committee, permission no.10/2018), and every effort was made to minimize the number of animals used for the experiments.

2.2.Preparation of Tissue Lysates

The lysates were obtained as described in [13]. The isolated structures were homoge-nized in lysis buffer (0.1 M Tris/HCl,2% SDS,50 mM DTT,pH8.0)and incubated for5min at 99 ℃.Samples were stored at-20 ℃ until proteomic analysis. Tryptophan fluorescence was employed to determine total protein concentration in the samples [14].

2.3.Multi-Enzyme Digestion Filter-Aided Sample Preparation(MED FASP)

The lysates containing 80 ug of total protein were used for the MED FASP[15] without alkylation of cysteine [16]. The proteins were cleaved overnight with LysC and then digested with trypsin for 3 h. The enzyme-to-protein ratio was 1:40. Digestions were carried out at 37 ℃ in 50 mM Tris-HCl with the addition of 1 mM DTI, pH 8.5 [13]Aliquots containing 8 ug of total peptide were concentrated and stored at -20 ℃ until mass spectrometry analysis.

2.4.Liquid Chromatography-Tandem Mass Spectrometry

The analysis of peptide mixtures was performed as described earlier[13] using the QExactive HF mass spectrometer (ThermoFisher Scientific, Palo Alto, CA, USA). The data were deposited in the ProteomeXchange Consortium via the PRIDE partner repository [17]. The dataset identifier: PXD025978(username: reviewer_pxd025978@ebi.ac.uk;password: QL3YI7nP).

2.5.Proteomic Data Analysis

MaxQuant v1.2.6.20[18] was used for the MS data analysis. The UniProtKB/Swiss-Prot database was employed to identify the proteins using MS and MS/MS peptide data.

Carbamidomethylation of cysteine was set as a fixed modification. cistanche dosage reddit The initial alowed mass deviation of the precursor ion was up to 6 ppm, and for the fragment masses, it was up to 20 ppm. The maximum false peptide discovery rate was specified as 0.01. The total protein approach method [19,20] was used to calculate the protein molar concentration using the relationship where the amount of individual proteins (c(i) was calculated as the ratio of their intensity (MSsignal(i))to the sum of allintensities(totalMSsignal) in the measured sample,multiplied by the molecular weight (MW(i)) of individual proteins.

2.6.Statistical Analysis

Data are presented as mean ± SD.The equality of variances was calculated using the Fisher F-test. To determine the differences between any two experimental groups, Student's t-test was used. The analysis was performed using SigmaPlot 11 software (Systat Software).

3. Results

The full proteomic data were deposited in the ProteomeXchange Consortium, and they are available with the dataset identifier: PXD0255978(username: reviewer_pxd025978@ebi.ac.uk;password: QL3YI7nP).The analysis of the spectra allowed for the quantitation of 7547 proteins across the analyzed samples. cistanche extract benefits Proteins that were identified with at least one unique pep-tide were used in the analysis. In detail, the expression data for7324,7088 and 7343 proteins in, respectively, the hippocampus, cortex and cerebellum of old animals were quantitatively determined and deposited to the ProteomeXchange Consortium via the PRIDE partner repository. For young animals, the data for7347,7323 and 7526 proteins in, respectively, the hippocampus, cortex and cerebellum were deposited therein.

4. Glutamatergic Transmission

Glutamate, the main excitatory neurotransmitter in the brain, acts through ionotropic and metabotropic receptors(mGluRs, Grm). The ionotropic receptors fall into one of four classes: o-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors(AMPA receptors, Gria), N-methyl-D-aspartate receptors(NMDA receptors, Grin), kainate receptors (Grik)and delta receptors (Grid). 4.1.Gria

Our study reveals that the most abundant glutamate receptors in the hippocampus are Gria. They are heterotetrameric proteins [21], and we found that the Gria2 subunit was expressed at the highest titer both in young and in aged murine hippocampi (Figure 1A). Aging had no effect on the expression of Gria receptors in the hippocampus, except for Gria4, whose level was significantly reduced in old animals. However, the titer of Gria4 was very low in general, as compared to other members of the Garcia family.

In the cerebral cortex, Gria were the most ubiquitous glutamate ionotropic receptors only in the old animals(Figure 1B,D). Statistically, the changes in the titers of individual Gria subunits during aging in the cortex were not significant, but the total Gria protein concentration was significantly increased in old animals (Figure 1B). cistanche genghis khan Similar to the hippocampus, Gria2 was the main isoform in the cortex.

In the cerebellum, the most abundant glutamate receptors were Grial and Gria2, which were expressed at very similar levels, and aging did not affect their concentration (Figure 1C). 4.2.Grin

NMDA receptors are glutamate-dependent heterotetrameric channels for calcium and sodium ions which are crucial for synaptic plasticity [2] and are composed of Grin isoforms [23]. We found that the main form of Grin expressed in all the studied brain structures was Grin1 (Figure 1A-C). The concentration of this isoform was not afected by aging in the hippocampus and cerebellum; however, it was significantly reduced in the cortex (Figure 1B). Overall, the total concentration of all NMDA subunits, except for Grin3a (whose level was unaffected by aging and expressed at a very low level as compared to other Grin proteins), was decreased in the aged cortex, while it was unchanged in the cerebellum (Figure 1B,C).In the hippocampus,aging was reflected by a statistically significant decrease in the total amount of Grin proteins, and also the Grin2b and Grin3a isoforms.

4.3.Grid

Gridl and Grid2 are ionotropic receptors expressed almost exclusively in the cere-bellum, and they are involved in the regulation of synaptic plasticity [24]. We found that in the cerebellum, the main isoform is Grid2, and that its titer is not afected by aging (Figure 1C).In the hippocampus and cortex, the concentration of, respectively, Gridl and Grid2 is significantly lower in aged animals; however, the titer of these receptors is very low in general (Figure 1A,B).

4.4.Grik

Kainate receptors are heteromeric sodium channels mediating ionotropic and metabotropic transmission [25,26]. Our analysis revealed that the concentration of almost all members of the Grik protein family was decreased in the old hippocampi and cortices (Figure 1A,B).In contrast, the titer of Grik proteins, except forthe Grik2 isoform, was unaffected by aging in the cerebellum(Figure 1C).

4.5.Grm

We identified seven members of glutamate-dependent metabotropic receptors, Grm, in all the studied brain formations: Grml-7 (Figure 1A-C).The total concentration of Grm proteins in the hippocampus and cortex was reduced in aged animals (Figure 1A,B), while the total titer of cerebellar Grms was not significantly changed by aging (Figure 1C).

Detailed analysis revealed that the levels of the most abundant isoforms of Grm (Grm2, Grm3, and Grm5) in the hippocampus were significantly decreased in old mice (Figure 1A), while in the cortex, the changes in the titers of the most abundant isoforms were not significantly altered (Figure 1B).

5.GABAergic Transmission

y-Aminobutyric acid is the most abundant inhibitory neurotransmitter in the brain, and it can interact with two types of receptors: the ionotropic GABAA receptor, which is a chloride channel, and the metabotropic GABAg receptor. GABA receptors are pentameric proteins composed of various subunits∶ a（Gabra），β（Gabrb），y（Gabrg）and δ（Gabrd）【27】GABAg is a heterodimeric G protein-coupled receptor formed by the Gabbrl and Gabbr2 subunits[28]. 6.GABAA-Gabr

Our analysis demonstrated that the total concentration of the GABA protein was significantly reduced in the hippocampus and cortex of old mice, but it was not afected by aging in the cerebellum (Figure 2A). These changes were reflected by alterations in the expression of individual subunits of the GABAA receptor(Figure 2B-D). 6.1.Gabra(subunit a)

We found that the overall concentration of Gabra subunits was not affected by aging in all the studied brain formations (Figure 2B-D). The most abundant isoform was Gabral. Within the a subunits, only Gabra3 and Gabra5 expression was influenced by aging (Figure 2B-D). The only statistically significant change was associated with Gabra4, whose titer was decreased in the cortex of aged animals (Figure 2C). 6.2.Gabrb(subunit β)

Our analysis showed that subunit β was the most ubiquitous GABAA subunit in all studied brain structures, both in young and in old animals (Figure 2B-D), and the main isoform of the β subunit was Gabrb2 (Figure 2B-D).The concentration of Gabr2b was the highest in the cerebellum, while in the hippocampus and cortex, its titers were similar (Figure 2B-D). We did not observe any significant aging-associated changes in the expression of isoforms of the β subunit in the hippocampus and cerebellum. However, in the cortex, the total titer of Gabrb was significantly reduced, and this was related to a decrease in the Gabrb2 titer (Figure 2C).

6.3.Gabrg（subunit γ）

Among the y subunits of the GABAA receptor, Gabrg2 was expressed at the highest level in the cortex and cerebellum (Figure 2C,D),and it was the only y subunit we could detect in the hippocampus (Figure 2B).In the hippocampus and cortex, the total Gabrg isoform concentration was almost two times higher in young animals than in aged ones (Figure 2A,B). On the other hand, the total y subunit concentration was not afected by aging in the cerebellum (Figure 2D). 6.4.Gabrd（subunitδ）

In old mice, the $ subunit was expressed mainly in the cerebellum (Figure 2B-D). The titer of Gabrd in the cerebellum of aged micewas about ten times higher than in the cortex, and more than 25 times higher than in the hippocampus (Figure 2B-D). Aging had no effect on Gabrd expression in the hippocampus and cerebellum, but in the cortex, the titer of the receptor was reduced more than three times by aging (Figure 2C). 7.GABAb-Gabbr

Gabbr is a heterodimeric metabotropic receptor formed by the Gabbrl and Gabbr1 subunits, and its function is coupled to activation of a G protein and modulation of activities of downstream effectors such as adenylate cyclase [29]. We found that the expression of both subunits of the metabotropic receptor was decreased in the hippocampus of the aged animals (Figure 2B).In the cerebellum, the Gabbr amount was unaffected by aging. We could also observe a significant reduction in the sum of the Gabbr subunit concentrations in the cortex, although separately, the changes in the titers of the subunits were not statistically significant (Figure 2C). 8.Gad

Gad is an enzyme decarboxylating glutamate to produce GABA [30]. Our data show that the expression of the main isoform of Gad in the hippocampus, Gad2, was not afected by aging; however, the level of another Gad isoform, Gad1, was significantly elevated (Figure 2B). We could also observe significant increases in the titers of both Gad isoforms in the cerebellum of aged animals (Figure 2D), but there were no changes in Gad expression in the cortex of old mice(Figure 2C).

9. Calcium/Calmodulin-Dependent Kinases

Activation of calcium/calmodulin-dependent protein kinase(Camk) is the first stage of transformation of calcium signaling into vaarious forms of synaptic plasticity(for review, see [31,32]). 9.1.Camk1

The only isoform of Camkl that we found in our analysis was Camkld. We also observed some Camkl-associated peptides, but we were not able to annotate them precisely to selected Camkl isoforms (they are described as"Camkl", Figure 3A-C).In our analysis, we did not observe any significant aging-associated changes in the concentration of that group of Camk in the hippocampus and cortex (Figure 3A,B).On the other hand, the titer of Camkl was significantly increased in the cerebellum of old mice (Figure 3C).

9.2.Camk2

Camk2 belongs to the most ubiquitous proteins in all brain structures [8,3]. We found that among Camk2 isoforms, the titer of Camk2a is the highest in all the studied brain structures (Figure 3A-D), and that its concentration in the hippocampus and cortex is many times higher than the titer of Camk2b, the second most abundant isoform of Camk2 (Figure 3A,B). Aging had no effect on the concentrations of the Camk2 isoforms, except for Camk2d, whose level was decreased in the hippocampus of old mice (Figure 3A).

9.3.Camk4

The molecular role of Camk4 in synaptic plasticity is different than Camk2. Active Camk4 localizes in the cell nucleus and regulates the transcription of genes involved in the late phase of memory formation [32].

Our study reveals that the concentration of Camk4 was significantly, almost two times, reduced in all the analyzed brain formations of old animals (Figure 3A-D). The protein titer was the lowest in the hippocampus and the highest in the cerebellum (Figure 3A-D).

9.4.Camkk

Calcium/calmodulin-dependent protein kinase kinases (Camkk) phosphorylate and regulate the activity of Camk proteins. Our analysis revealed that the total concentration of Camkk was not statistically significantly affected by aging in the hippocampus and cortex (Figure 3A,B,D).In the cerebellum, the level of the Camkkl isoform was more than 3-fold increased in old animals, whereas the Camkk2 concentration was more than two times lower in aged animals (Figure 3C).

10.Prka-cAMP-Dependent Protein Kinase (PKA)

Prka is a conserved serine protein kinase with a wide distribution and relatively low specificity, which can phosphorylate various subunits of the AMPA and NMDA receptors and modulate their function [34]. 10.1.PKA Catalytic Subunits—Prkac

Our analysis demonstrated that in the hippocampus, the most abundant catalytic subunits of PKA are Prkaca and Prkacb (Figure 4). The concentrations of individual subunits were not affected by aging; however, the totalPrkac protein concentration was slightly but statistically significantly decreased in aged mice (Figure 4A,D). Similar to the hippocampus, in the cortex and cerebellum, the most ubiquitous catalytic subunits of PKA were Prkaca and Prkacb; however, the concentrations of the proteins did not difer between young and old animals(Figure 4B,C). 10.2.PKA regulatory subunits——Prkar

We found that the overall expression of Pekar isoforms was the highest in the cortex, while in the hippocampus and cerebellum, the level of Prkar was similar (Figure 4A-D). Aging had no effect on the total concentratiorn of Prkar, and the only age-related difference was a reduced level of Prkar2b in the hippocampus of old mice (Figure 4A).

11. Mitogen-Activated Protein Kinases—Mapk

Mark proteins regulate a broad spectrum of cytoplasmic and nuclear processes involved in neuronal plasticity [35].

The results presented here demonstrate that Maple was the most abundant Mapk in the hippocampus (Figure 4A).The total Mapk concentration in the hippocampus was not affected by aging (Figure 4A,D);however,Mapk3 expression was about 23% higher in aged animals (Figure 4A), and Mapk15 was increased almost seven times in aged hippocampi (Figure4A).

Both in the cortex and in the cerebellum, the total Mapk concentration was not affected by aging, and Mapk1 was the main kinase expressed in the two brain structures (Figure 4B,C).

This article is extracted from Cells 2021, 10, 2021. https://doi.org/10.3390/cells10082021 https://www.mdpi.com/journal/cells